Amyotrophic lateral sclerosis (ALS, also known as Lou Gehrig's disease) is a fatal neurological disease. In patients with ALS, degeneration and death of cortical and spinal motor neurons leads to progressive muscle paralysis, often starting in the distal limbs and progressing to the respiratory muscles (Cleveland and Rothstein, 2001). The number of new ALS cases per year worldwide is about 125,000. However, the sole approved drug, riluzole, confers only minor benefit. There is still no effective therapy for the adult-onset neurodegenerative disease ALS (amyotrophic lateral sclerosis). One major explanation is that very few therapeutic targets have been validated.
Therapeutic targets in ALS are rare. One of the major obstacles to a successful therapy for ALS is the near-absence of validated targets, molecular events in the disease pathway whose inhibition slows onset or progression. Genes such as superoxide dismutase 1 (SOD1) whose mutation can lead to ALS may be considered to be validated targets, but familial forms of the disease collectively represent only 10% of all cases. Therapeutic targets applicable to the 90% of sporadic cases would likely be key downstream effectors of the disease pathway, but no such intermediates have been validated. If genes acting early in the pathway can be identified, they will provide a solid foundation for targeted drug discovery programs.
Target discovery in mouse and human models of ALS. A great majority of studies on the mechanisms of ALS have focused on mouse models expressing disease-triggering mutant forms of SOD1 (Turner and Talbot, 2008). These mice develop a disease that is in many respects a close reflection of not only familial but also sporadic ALS, including selective resistance of oculomotor and slow spinal motor neurons (Valdez et al., 2012). However, even in this model, there are few validated targets other than the SOD1 gene itself. Moreover, concerns have been raised that results obtained in mSOD1 mice may not translate well to the clinic. Although this may in part reflect underpowered mouse preclinical studies, there is a need for human models to discover and validate novel candidate disease modifiers.
Although the disease mechanism involves other cell types, motor neuron dysfunction and degeneration are the defining features of the clinical phenotype, and cell-autonomous deficits in motor neurons are known to contribute to disease onset. It remains to be determined, however, how ALS motor neurons differ from their counterparts in healthy individuals at early stages of the disease, and this has hindered the development of assays for disease-modifying drugs. Strikingly, none of the many drugs that have undergone clinical trials in ALS was ever tested on the cells affected in the disease: sick human motor neurons. A reasonable strategy would be to evaluate candidate treatments in two systems in parallel: human motor neurons in vitro and the mouse neuromuscular system in vivo.
For this reason, the inventors have sought to use human induced pluripotent stem cells (hiPSCs) to model ALS in the culture dish. Multiple iPS lines from ALS patients and controls have been successfully generated and shown that they can be robustly differentiated in culture into motor neurons (iPS-MNs) and other spinal cord cell types such as astrocytes (Boulting et al., 2011; Dimos et al., 2008). However, to date, no spontaneous ALS-related phenotype has been reported in such cultures, perhaps because ALS is an adult-onset disease. Therefore, there is a need to better understand the molecular and functional differences between ALS and control iPS-MNs and to use this knowledge to identify disease-relevant neuroprotective agents.
Contribution of motor neuron hyperexcitability and Ca++ imbalance to the ALS phenotype. Neuronal excitability reflects the critical balance of activation among dozens of membrane channels and their binding partners. Changes in the activation or inactivation properties of even a single channel can dramatically alter excitability of a single neuron or network of neurons. Any increase in membrane excitability ultimately results in elevations in intracellular Ca++ concentration which, when excessively sustained, can result in cell death. Specific increases in motor neuron excitability have been seen in neonatal ALS model mice, months before the onset of overt clinical symptoms (Quinlan et al., 2011). These changes seem to reflect alterations in intrinsic properties of motor neurons, because they can be observed in isolated embryonic neurons in culture. However, the gene differences underlying this hyperexcitability remain to be determined. If targets could be identified to modify motor neuron excitability, their modulation would potentially have early and potent effects.
More generally, calcium dysregulation is a frequently observed defect in models of ALS (reviewed in Grosskreutz et al., 2010). There is evidence from multiple sources for involvement of excitotoxicity, excessive Ca++ influx through ionotropic glutamate receptors. This can result from defective glutamate reuptake, altered Ca++ permeability of AMPA receptors by changes in subunit composition or RNA editing. Another site of calcium dysregulation is in mitochondria, which are strongly affected in the disease. In ALS models, mitochondria appear to be less able to handle large Ca++ loads induced by electrical activity, leading to chronic Ca++ overload. Lastly, the endoplasmic reticulum (ER) shows pathological changes at morphological and functional levels. ER stress is directly triggered by disease-causing mutations in vesicle-associate membrane protein-associated protein B (VAPB) and also by the unfolded protein response (UPR) observed in cells expressing misfolded mutant forms of SOD1. The shuttling of Ca++ between the ER and mitochondria, called the ER-mitochondria Ca++ cycle (ERMCC), is therefore a potentially important site of dysfunction in ALS. However, much remains to be learned of the ways in which it is specifically affected in ALS motor neurons.
Accordingly, there is, inter alia, a need to identify agents for the treatment and amelioration of ALS. The present invention is directed towards meeting this and other needs.